Resampling and Editing of Mischarged tRNA Prior to Translation Elongation

Abstract

SUMMARY Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu•GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing pathway, leading to a reduction in the overall error rate of aminoacyl-tRNA synthesis. Resampling of mischarged tRNAs was shown to increase the accuracy of translation over ten fold during in vitro protein synthesis, supporting the presence of an additional quality control step prior to translation elongation.

Homologous trans -editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation

“…Product release is rapid in class II ARSs, unlike class I enzymes in which this step is rate limiting (41). Thus, class I ARSs remain bound to their substrates long enough to edit any misacylated tRNA before release and normally would not require proofreading by a trans-editing factor (42). In the assay demonstrating ThrRS protection, we also expect that class II ThrRS releases the substrate rapidly; however, ThrRS competes effectively with ProXp-ST1 and -ST2 in rebinding the cognate Thr-tRNA Thr substrate and therefore protects it from hydrolysis.…”

Kinetic Partitioning between Synthetic and Editing Pathways in Class I Aminoacyl-tRNA Synthetases Occurs at Both Pre-transfer and Post-transfer Hydrolytic Steps

“…A trans-editing model has also been proposed for class II aaRSs. In this model, the misacylated tRNA is released from the enzyme but then undergoes hydrolysis after rebinding (15).…”

“…Although this pathway is naturally suppressed by fast deacylation of misacylated tRNA within the CP1-editing site in WT LeuRS, it may nonetheless represent a mopping up activity for any misacylated tRNAs that escape cis editing. A dissociation-reassociation pathway by which misacylated tRNAs are released and rebound by the aaRS editing domains has been proposed as a significant editing pathway for class II aaRSs (15). Explicit measurements of association and dissociation rates for cognate and noncognate aa-tRNAs for class I editing enzymes will offer more quantitative insight into this phenomenon.…”

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

“…Following this process, the aminoacyl-tRNA is released from the synthetase and bound by elongation factor Tu (EF-Tu) 2 for delivery to the ribosome and use in protein synthesis (6,7). Aminoacyl-tRNA synthetases are usually highly selective for their cognate tRNA due to the availability of a large surface area for recognition, identity elements within the tRNA molecule itself, and also kinetic proofreading during the aminoacylation reaction (8 -10).…”